LISTED UNDER:

Figure 1: a) Perfinity Workstation integrates necessary steps for immuno-MS workflows. Immunoaffinity enrichment of hemoglobin alpha (HbA) subunit was carried out using a Perfinity Workstation. Online enrichment and subsequent digestion and LC-MS/MS using a Shimadzu IT-TOF showed three characteristic peptides from HbA. The optimized affinity-to-peptide method was carried out in less than 45 min from injection to detection. b) TIC of affinity enriched and digested HbA. c) Extracted ion chromatograms for surrogate peptides of HbA with MS/MS confirmation example for peptide VGAHAGEYGAEALER. Image: Shimadzu Scientific Instruments

Progress often requires change. For protein-based diagnostics, multiplexed assays and detection of protein isoforms will drive the adoption of a new strategy for diagnostic testing, called “immuno-MS”.

Enzyme-linked immunosorbent assays (ELISA) have become the standard for antibody-based diagnostic tests in clinical settings. ELISAs provide specific detection of biomarkers through use of antibodies which target specific epitopes on the antigens. Proteins often have many different isoforms which may differ by a single amino acid or a post-translational modification. ELISAs generally don’t have the ability to differentiate the presence and quantity of isoforms as has been shown in the example of prostate specific antigen (PSA). With PSA, it’s not the protein itself, but the different glycoforms of PSA which have been shown to be the diagnostic markers. An additional challenge faced with ELISAs has been the ability to multiplex, or to detect multiple antigens in a single assay.

Immuno-purification combined with chromatographic separations and mass spectrometry, also known as immuno-MS or mass-linked immuno-selective analysis (MALISA), may provide a solution for limitations associated with ELISAs. Cho et al. illustrated that on column immuno-precipitation using an anti-sialyl lewis x IgM antibody in line with reversed phase chromatography had the ability to differentiate between a stage-2 ductal carcinoma plasma and NIST normal pooled plasma. In this workflow, the anti-sLex enrichment antibody provided specificity to pull out of solution a collection of proteins bearing the sLex motif. Furthermore, the reversed phase provided the resolution to separate the proteins.

By combining two orthogonal separations, nine Lewis x (Lex)- or sLex-containing proteins were found to be differentially expressed in breast cancer patients. The possibility to multiplex these nine biomarkers in a single assay using affinity enrichment and LC-MS opens up opportunities not previously available by ELISAs.

Sample preparation for immuno-MS workflows generally involves affinity enrichment, buffer exchange, proteolysis, de-salting, drying, re-suspending and reversed phase chromatography. These disjointed, user-dependent protocols generally lead to highly variable results and can take more than 24 hrs to complete. For example, Becker et al. describe an LC-MS/MS CISCAPA workflow that takes slightly over two days. The bulk of the protocol is spent on digestion and forming the immune-complex, indicating that sample preparation is a bottleneck for immuno-MS protocols. Additionally, the authors comment that trypsin digestion rarely goes to completion and contributes to a significant amount of variability. With digestion as an indispensable step in immuno-MS workflows, improving throughput and reproducibility is critical.

click to enlargeFigure 2: a)The noviplex card is a sample collection device that can separate whole blood into a fixed volume of plasma (2.5 or 5.0 μL) in 3 min without use of centrifuges. b) An amount of blood is applied to the test area (20 to 75 μL) until the control spot turns red. c) The sample dissipates across a spreading layer, the hemotocrit is removed and plasma is collected on a removable disc. d) After three minutes, the cover is peeled back. The collection disc is dried for 15 minutes at which point it can be stored, transported or analyzed.

It was previously discussed how the Perfinity integrated Digestion Platform (iDP) (Perfinity Biosciences, W. Lafayette, Ind.) can provide reproducible digestions in minutes through automation, optimized buffers and trypsin immobilized enzyme reactors (IMERs). The Perfinity Workstation expands on the iDP by including immunoaffinity enrichment and buffer exchange prior to digestion.

Figure 1a illustrates the workflow for the Perfinity Workstation. In brief, antibodies are spiked into the sample to form the immune complex. The spiked sample is then injected onto the workstation where an affinity column pulls the immune complex out of solution. Following trapping, the interaction of the immune complex is disrupted with acidic conditions, and released proteins pass through a buffer exchange column where the pH is brought up to optimal conditions for trypsin digestion. The sample is then rapidly digested in approximately six minutes, de-salted and separated by reversed phase chromatography.

To illustrate the capabilities of the Perfinity Workstation, Hemoglobin subunit alpha (HbA) was enriched using an Anti-HbA column. Captured HbA was rapidly digested in six minutes, de-salted and analyzed by LC-MS/MS (Figure 1b and c). The total analysis time (immunocapture, buffer exchange, trypsin digestion, de-salting and reversed phase) on the workstation was 45 min.

Resulting coefficients of variance (CVs) for the digestion protocol with trypsin IMERs have been shown to be typically 3 to 5%, with a maximum around 10%. The improvement in reproducibility can be attributed to making digestion and de-salting a streamlined, hands-free protocol. Additionally, the trypsin IMER increases the ratio from a 1:20 protease-to-protein ratio to 1000:1 protease-to-protein ratio. The drastic increase in trypsin concentration leads to more interactions between the proteins and proteases, thus decreasing the digestion time from overnight to minutes.

In the future, lab-on-a-card technology will enable immune capture at the collection site, even further simplifying and accelerating immuno-MS workflows.

A new plasma collection device for dried plasma spot analysis (DPS), the Noviplex card (Novilytic LLC, North Webster, Ind.) provides easy collection of plasma from small volumes of blood such as fingerpricks or mouse tail bleeds. Figure 2 illustrates the workflow for the Noviplex card. These self-contained sample collection cards have the ability to generate a fixed volume of plasma (2.5 µL or 5.0 µL) from small volumes (20 µL to 75 µL) without the use of centrifuges. In only three minutes, capillary action pulls the sample through a series of layers, first spreading the sample, then removing the hematocrit before the fixed volume of plasma is collected. Functionalized Noviplex cards will have the ability to capture proteins of interest creating an assay-ready sample immediately upon sample collection.